1. Field of the Invention
The present invention relates to an apparatus for rapid screening of chiral molecules, to method for making the apparatus and to methods for using the apparatus to screen chiral molecules, especially, catalyst, bio-catalyst, bio-molecules such as polypeptides, proteins, enzymes, ribozymes, or the like or mixtures or combination thereof.
More particularly, the present invention relates to an apparatus including a sample cell, a polarized light source for supply polarized light to a sample in the cell, a magnetic field generator surrounding the cell for applying a time varying magnetic field to a sample in the cell, an analyzer for analyzing the transmitted light from the sample, a detector for detecting the output of the analyzer and generating a sample output signal, a lock-in amplifier for receiving the detector output signal and generating an amplitude and a phase of the detector output signal and a power supply for supplying a time varying electrical input to the magnetic field generator and for supplying a reference input or lock input to the lock-in amplifier. The present invention also relates to methods for making and using the apparatus.
2. Description of the Related Art
Many diseases are still without an effective treatment today, and others have evolved resistances requiring new therapeutic agents to be developed. Among these ailments are the neurodegenerative diseases, autoimmune diseases, cancer, viral diseases, fungal infections, tropical parasitic diseases, in addition to emerging antibiotic resistant pathogenic bacterial strains. Treatment of many such diseases will depend on inhibiting a key enzyme or enzymes. Examples include: cathepsin D as a novel treatment for Alzheimer's7, matrix metalloproteases (MMPs) for rheumatoid arthritis50 and early stage cancers33,70, HIV integrase13, HIV protease48,. HIV reverse transcriptase49 in the cases of AIDS, cytochorome P450 enzyme lanosterol 14α-demethylase for fungal infections12,65, glutathione reductase for the treatment of Malaria40, cruzain and rodesian for the treatment of the trypanosomal diseases Chargas disease, African sleeping sickness, and Nagana19, and new antibiotics for targeting bacterial cell wall synthesis, protein synthesis, and DNA replication14.
Fortunately, the advent of combinatorial chemistry has helped augment the pharmaceutical industries' capacity to develop new lead compounds to treat these diseases. Coupled with the recent advances in bioinformatics for identifying new targets, there exists tremendous potential for developing effective small-molecule therapeutic agents, not only for these diseases, but also for those ailments for which no effective treatment exists21.
In the effort to develop these discovery candidates for medical applications, an important observation is that the vast majority of useful drugs contain one or several chiral centers55. The wrong enantiomer can unfortunately cause harmful side effects, so very high enantiomeric purity of therapeutics is also essential12. The observation that chirality can play a major role in the toxicity and specificity of therapeutic agents was first made in 1956 by Carl Pfeiffer45 and is commonly referred to as Pfeiffer's Rule which states “. . . the greater the difference between the pharmacological activity of the D and L isomers the greater is the specificity of the active isomer for the response of the tissue under test.” Thus, both producing enantiomerically pure formulations and testing for enantiomeric purity are critical. Unfortunately, both of these activities remain significant challenges, even with the current state-of-the-art analytical instrumentation.
The realization that enantiomeric purity plays a critical role in the specificity and toxicity of pharmacological agents has prompted the Food & Drug Administration to increase its regulatory oversight into enantiomeric purity of approved pharmaceuticals. Recent FDA policy71 requires that any drug component over 1% of total composition be tested with separate toxicology studies. Thus a racemic drug candidate would require separate trials for each enantiomer. While enantiomeric purity is not mandated by the FDA, the huge cost of clinical trials virtually ensures that pharmaceutical companies will develop enantiomerically pure drug candidates,56 provided the enantiomers do not readily interconvert.
A variety of process solutions, such as resolution of racemates or asymmetric synthesis, are utilized by the pharmaceutical industry to provide these enantiomerically pure drugs. Owing to lack of suitable catalysts or insufficient selectivity, however, efficient enantioselective synthesis of these enantiomers remains a major challenge. As a result, resolution methods still dominate in industrial production56. This limits their transfer from therapeutic concept to cost-effective drugs on the market.
While recent progress in the area of biocatalysis to provide more stereoselective catalysts is encouraging44, the number of enzymes commercially available remains limited. However, advances in the area of directed evolution and metabolic engineering show promise of changing this situation68. In some special cases, where screens have been established for specific substrate enantiomeric purity, directed evolution has proven to be capable of improving enantioselectivity of enzymes. Unfortunately these screens require expensive or modified reagents, and have only moderate (thousands/day) throughput41. Therefore not all compounds of interest are amenable to these approaches, and the low throughput limits the true utility of the directed evolution approach4. The same limitation applies to non-enzymatic combinatorial chemistry approaches, where large libraries must be screened for activity to determine successful chiral catalyst22 or screening environmental samples for novel activity64. “You get what you screen for”3 and to date no generally applicable method for high throughput enantiomeric purity screening is available to the researcher.
As, by definition, chiral molecules display optical rotation (they rotate the polarization of light, a property that is also called “optical activity”), polarimetry would seem to be an excellent technique for search and optimization of efficient enantioselective catalysts. However, polarimetry currently lacks several features essential for advantageous utilization in the development of chiral catalysts. Such features include sensitivity: the best polarizers are constructed from material (calcite) that cannot be grown in the laboratory. So naturally occurring calcite are commonly used, but it has imperfect optical quality and hence results in limited sensitivity. Also, polarimetry is one-dimensional, yielding only one parameter (the optical rotation) and hence is a weaker indicator than one would like: a multi-parameter result would be much more useful. Another needed feature is the ability to perform high-throughput screening: the best polarimeters utilize the highest quality calcite crystals, which allow only one sample to be measured at a time due to their small size (<20 mm×20 mm). In addition, current polarimeters require sample chambers with long path lengths (100 mm), relatively high sample concentrations (mg/mL), and long data-acquisition times, which allow only ten to twenty samples per hour. While larger dichroic sheet polarizers have been used to allow the screening of ˜100 wells simultaneously by imaging24,25,66, this technique lacks sensitivity due to the rather poor extinction coefficient (the ratio of light transmitted when the polarizers are crossed to that when they are parallel, an indicator of polarizer quality) of sheet polarizers.
Thus, there is a need in the art for a next-generation polarimeter apparatus and method that overcomes all these problems and which is suitable for rapid, accurate, and large-scale screening of catalysts or other chiral compounds including pharmaceuticals.